Spinal Muscular Atrophy (SMA) is a leading genetic cause of infant mortality. Most commonly, SMA results from the reduced levels of full-length SMN protein (SMN) in motor neurons and spinal chord due to the loss of functional Survival Motor Neuron (SMN1) alleles. A nearly identical copy of this gene, SMN2, fails to provide protection from SMA due to production of a truncated SMN because of skipping of SMN2 exon 7 during pre-mRNA splicing. There is a near consensus among researchers that strategies aimed at promotion of SMN2 exon 7 inclusion resulting into the increased levels of full- length SMN would cure SMA. Towards this goal we have recently reported an eight-nucleotide GC- rich intronic target, sequestering of which by an antisense oligonucleotide (ASO) fully restored SMN2 exon 7 inclusion in SMA patient cells. Specificity and efficiency of antisense response by our 8-mer lead ASO (3UP8) targeting GC-rich sequence constitute the first such report of splicing correction by a short ASO in a patient cell line. The unmatched benefits of a short ASO as a therapeutic agent include but not limited to the expected high specificity, low cost of synthesis, ease of modifications and increased chances of delivery across biological barriers. Currently SMA has no cure. As one of the best hopes of SMA therapy, here we propose to develop an optimized variant of our lead ASO that efficiently corrects SMN2 exon 7 splicing and raise the levels of full-length SMN in all tissues including brain and spinal cord of mice models of SMA. In Aim 1, we will optimize our short ASO for application in vivo. These will be accomplished by a series of custom modifications including different combinations of the terminal and backbone chemistries. We will test the efficacy of modified short ASOs in transgenic mice containing human SMN2. Our initial results validate the proof-of-principle that custom modifications improve the efficacy of a short ASO in splicing correction in vivo. We will run a series of tests to confirm the in vivo efficacy of the custom-modified ASOs. These include but not limited to stability, dose response, off-target effects, immune response and pharmacokinetic properties. We believe that a combination of chemical modifications will allow us to obtain an optimized lead ASO that could be effectively delivered in all tissues including brain and spinal cord. Will also test an alternatively lipid-nanoparticle-based approach to efficiently deliver our optimized lead ASO in different tissues including across BBB. In Aim 2, we will perform experiments in mild as well as severe SMA mouse models. We will determine the effect of different doses of our optimized lead ASO on the phenotype, growth and development of SMA mice. In particular, we seek to improve the longevity of SMA mice. We will also conduct experiments in pregnant mice to see the effect of pre-natal drug delivery on the phenotype and longevity of SMA offspring. The success of this proposal will provide a mechanism-based and target- specific drug for the treatment of SMA. ) PUBLIC HEALTH RELEVANCE: This project will develop and test the efficacy of short antisense oligonucleotides for correction of aberrant splicing in mice models of spinal muscular atrophy (SMA). The success of this proposal will lead to the treatment of SMA, a leading genetic cause of infant mortality. )